Enhancing algal biomass and biofuels recovery from open culture

194 Pages·2015·5.65 MB·English

by  Lisa Bai

Enhancing algal biomass and biofuels recovery from open culture
systems
Xue Bai

A thesis submitted for the degree of Doctor of Philosophy at
The University of Queensland in 2015
School of Chemical Engineering

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Abstract
Growth of algae for the purposes of generating biofuels and/or other bioproducts holds promise due
to numerous advantages unique to algae. But there are serious barriers to commercial scale algae
cultivation and downstream processing. Two barriers addressed in this thesis are: (i) carbon limitation
in open pond algal cultivation, and (ii) difficulties for downstream processing due to the robust nature
of algal cells. Hence the overall aims of this thesis were to:
 Understand and model the contribution of carbon cycling by heterotrophic bacteria to algal
growth, and
 Facilitate effective downstream processing by applying a novel free nitrous acid (FNA) pre-
treatment to disrupt algal cells.
To understand the contribution of heterotrophic bacteria to algal growth, carbon cycling driven by
bacteria was evaluated in open algal culture systems. Since pH controls the proportion of carbon in
the various inorganic pools, two different pHs were trialled. The contribution of bacteria to carbon
cycling was determined by quantifying algae growth with and without supplementation of bacteria.
It was found that adding heterotrophs to an open algal culture dramatically enhances algae
productivity. Increases in algae productivity due to supplementation of bacteria of 4.8 and 3.4 times
were observed in two batch tests operating at two different pH values over 7 days. A simplified kinetic
model was proposed which described carbon limited algal growth. Simulation of the model
highlighted how carbon limitation can be overcome by bacteria re-mineralising photosynthetic end
products. The model was extended into a full Algae-Bacteria Model (ABM) to describe carbon,
oxygen and nitrogen flows in open algal systems. The integrated model, presented in an easy to use
Petersen’s matrix format, provides the first description of algal cultivation processes considering the
effect of heterotrophic bacteria driven carbon cycling.
The second aim of the thesis was addressed through the development and use of a novel pre-treatment
technology – FNA pre-treatment – to enhance biofuels production, including (i) enhancing lipids, and
specifically triacylglycerides (TAG) recovery for biodiesel production, and (ii) enhancing methane
yield during anaerobic digestion of algae.
For enhancing lipids, and specifically TAG recovery, laboratory batch tests, with a range of FNA
conditions and pre-treatment time, were conducted to disrupt algal cells prior to lipid extraction by
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organic solvents. Total lipid (TL) quantified by the Bligh and Dyer method was found to increase
with longer pre-treatment time (48 h) and higher FNA concentration (up to 2.19 mg HNO -N L-1).
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Lipid extraction kinetic analysis was also conducted by Soxhlet extraction apparatus. The mass
transfer coefficient (k) for lipid extraction from algae treated with 2.19 mg HNO -N L-1 FNA was
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found to increase dramatically to 0.96 h-1 over the untreated algae (0.39 h-1). The quantity of TAG
among total lipid recovered was boosted with increasing FNA pre-treatment up to an FNA
concentration of 2.25 mg HNO -N L-1. But higher FNA concentrations (13.49 and 26.98 mg HNO -
2 2
N L-1) were detrimental to polyunsaturated fatty acid (PUFA) recovery, which resulted in a decrease
in TAG recovery.
For enhancing methane yield during algal digestion, the feasibility of using FNA pre-treatment was
investigated through laboratory biochemical methane potential tests. It was demonstrated that
methane production was dramatically enhanced by FNA pre-treatment (2.31 mg HNO -N L-1), with
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a 53 % increase in the methane yield (from 161 to 250 L CH per kg VS). A two substrate model was
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used to describe digestion to account for the apparent presence of rapid and slowly degradable
material. Model-based analysis revealed that with FNA pre-treatment, the availability of both rapid
and slowly biodegradable substrates were increased.

ii
Declaration by author
This thesis is composed of my original work, and contains no material previously published or written
by another person except where due reference has been made in the text. I have clearly stated the
contribution by others to jointly-authored works that I have included in my thesis.
I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance,
survey design, data analysis, significant technical procedures, professional editorial advice, and any
other original research work used or reported in my thesis. The content of my thesis is the result of
work I have carried out since the commencement of my research higher degree candidature and does
not include a substantial part of work that has been submitted to qualify for the award of any other
degree or diploma in any university or other tertiary institution. I have clearly stated which parts of
my thesis, if any, have been submitted to qualify for another award.
I acknowledge that an electronic copy of my thesis must be lodged with the University Library and,
subject to the General Award Rules of The University of Queensland, immediately made available
for research and study in accordance with the Copyright Act 1968.
I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s)
of that material. Where appropriate I have obtained copyright permission from the copyright holder
to reproduce material in this thesis.

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Publications during candidature
Xue Bai, Paul A. Lant, Steven Pratt (2014). The contribution of bacteria to algal growth by carbon
cycling. Biotechnology and Bioengineering. doi: 10.1002/bit.25475.
Xue Bai, Forough Ghasemi Naghdi, Liu Ye, Paul A. Lant, Steven Pratt (2014). Enhanced lipid
extraction from algae using free nitrous acid pre-treatment. Bioresource Technology. 159: 36-40.
Xue Bai, Peer M. Schenk, Zhiguo Yuan, Paul A. Lant, Steven Pratt (2015). Enhanced triacylglyceride
extraction from microalgae using free nitrous acid pre-treatment. Applied Energy. (accepted subject
to revision).
Xue Bai, Paul A. Lant, Paul D. Jensen, Sergi Astals, Steven Pratt (2014). Enhancing methane
production from algae by free nitrous acid pre-treatment (Submitted).
Jessica J. Buchanan, Frances R. Slater, Xue Bai, Steven Pratt. (2012). Algal growth and community
structure in a mixed-culture system using coal seam gas water as the water source. Environmental
Technology. 34(6): 695-701.

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Publications included in this thesis
Xue Bai, Paul Lant, Steven Pratt (2014). The contribution of bacteria to algal growth by carbon
cycling. Biotechnology and Bioengineering. doi: 10.1002/bit.25475.
X Bai operated all the experiments. P Lant, and S Pratt advised on the experimental design. P
Lant and S Pratt critically reviewed the paper.
The research objectives, approach and outcome of this paper are included in Chapter 3-5 and
the paper is wholly attached in the Appendix A.
Xue Bai, Forough Ghasemi Naghdi, Liu Ye, Paul Lant, Steven Pratt (2014). Enhanced lipid extraction
from algae using free nitrous acid pretreatment. Bioresource Technology. 159: 36-40.
X Bai operated the experiments of algae cultivation and harvesting, free nitrous acid (FNA) pre-
treatment and Bligh and Dyer lipid extraction. X Bai and F Ghasemi Naghdi conducted the
Sohxlet lipid extraction experiments jointly. L Ye, P Lant, and S Pratt advised on the experimental
design. P Lant and S Pratt critically reviewed the paper.
The research objectives, approach and outcome of this paper are included in Chapter 3-5 and
the paper is wholly attached in the Appendix C.
Xue Bai, Peer M. Schenk, Zhiguo Yuan, Paul A. Lant, Steven Pratt (2015). Enhanced triacylglyceride
extraction from microalgae using free nitrous acid pre-treatment. Applied Energy. (accepted subject
to revision).
X Bai operated the experiments of algae cultivation and harvesting, free nitrous acid (FNA) pre-
treatment and total lipid extraction. P Schenk supported for fatty acid profile characterisation.
Z Yuan, P Schenk, P Lant, and S Pratt advised on the experimental design and critically reviewed
the paper.
The research objectives, approach and outcome of this paper are included in Chapter 3-5 and
the accepted paper is wholly attached in the Appendix D.
Xue Bai, Paul Lant, Paul Jensen, Sergi Astals, Steven Pratt (submitted 07/2014). Enhancing methane
production from algae by free nitrous acid pre-treatment. Renewable Energy.
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X Bai operated the experiments of algae cultivation and harvesting, free nitrous acid (FNA) pre-
treatment and biochemical methane potential tests. X Bai and S Astals conducted the two-
substrate model analysis. P Lant, P Jensen and S Pratt advised on the experimental design and
critically reviewed the paper.
The research objectives, approach and outcome of this paper are included in Chapter 3-5 and
the submitted paper is wholly attached in the Appendix E.
Contributions by others to the thesis
This thesis includes contributions made by others, particularly in the chemical analysis of wastewater
and reactor samples. These contributions are acknowledged as follows:
Dr. Beatrice Keller-Lehmann, Jianguang Li and Nathan Clayton operated Flow Injection Analyzer
(FIA) for ammonia, nitrate, nitrite, and phosphate detection and the Total Organic Carbon Analyser
for total inorganic carbon (TIC) detection.
Statement of parts of the thesis submitted to qualify for the
award of another degree
None.

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Acknowledgements
I would like to thank my advisors Prof. Paul Lant and Dr. Steven Pratt, for their kindness, patience
and most important academic excellence. I am grateful for their guidance, ever available to provide
feedback and to have discussions with me, and for always having the faith in me to develop and
explore my ideas. Both of their enthusiasm for research has been always an inspiration and their
ability to structure and refine documents impressed me every time. Steven was always available to
talk with me with my problems; and Paul helped me to step back to view the whole picture and
provide me intelligent advice in many different aspects. I am sincerely grateful for the opportunity to
work with them.
I would also like to thank Prof. Zhiguo Yuan and Dr. Liu Ye for their promotion of FNA techonogy
for cell disruption. I am also grateful to Prof. Peer Schenk who provided valuable insights and
research resources to my research. I thank the staff at the School of Chemical Engineering and AWMC
for helping with all administrative and analytical tasks.
I am grateful to the students who I have worked with in my project. Philip, who is always willing to
discuss with me and helped me finding my way out. Forough, who is more than a good friend to me,
was always available to help me with my problems; both those relating to the PhD and personal ones.
A big thank you to Monica, Liliana, Wei and the people from Prof. Schenk’s lab, who are always open
to discuss and talk.
It has been a pleasure for me working with the people down in Chem Eng PhD room. You have been
a great group of friends who have helped me to get through the thesis by creating such an enjoyable
working environment. It has been a pleasure working with you and I can only hope that my next
workplace will be full of people as great as you.
I am also grateful to the financial support from China Scholarship Council and the School of Chemical
Engineering for topping me up the scholarship.
Importantly, I would give a big hug to my dear parents who are in China but never stopped giving me
love and support. I would also like to thank my friends who are far away and scattered around
Australia and the world but have never failed to send their love and support.
Now I am finally at the end of the PhD track. Another journey just begins.
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Keywords
algae, bacteria, model, free nitrous acid, lipid, triacylglyceride, anaerobic digestion, methane

Australia and New Zealand Standard Research
Classifications (ANZSRC)
100302 Bioprocessing, Bioproduction and Bioproducts, 30 %
090703 Environmental Technologies, 30 %
090499 Chemical Engineering not elsewhere classified, 40 %

Fields of Research (FoR) Classification
0904 Chemical Engineering, 100 %

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Table of contents
Abstract ........................................................................................................................................... i
Declaration by author ..................................................................................................................... iii
Publications during candidature ..................................................................................................... iv
Publications included in this thesis .................................................................................................. v
Contributions by others to the thesis ............................................................................................... vi
Statement of parts of the thesis submitted to qualify for the award of another degree ...................... vi
Acknowledgements ....................................................................................................................... vii
Keywords ..................................................................................................................................... viii
Australia and New Zealand Standard Research Classifications (ANZSRC) .................................. viii
Fields of Research (FoR) Classification........................................................................................ viii
Table of contents ............................................................................................................................ ix
Nomenclatures ............................................................................................................................... xi
List of Figures .................................................................................................................................. i
List of Tables ................................................................................................................................. iii
1 INTRODUCTION ................................................................................................................. 1
1.1 Background ................................................................................................................... 1
1.2 Thesis Objectives – General .......................................................................................... 1
1.3 Thesis Outline ............................................................................................................... 2
2 LITERATURE REVIEW ....................................................................................................... 3
2.1 Introduction ................................................................................................................... 3
2.2 Current technologies and opportunities for process enhancement ................................... 5
2.2.1 Algal cultivation .............................................................................................. 7
2.2.2 Harvesting ..................................................................................................... 10
2.2.3 Pre-treatment ................................................................................................. 11
2.2.4 Lipid extraction ............................................................................................. 12
2.2.5 Biodiesel production...................................................................................... 13
2.2.6 Anaerobic digestion and biomethane production ............................................ 14
2.3 Open algal culture systems (Part I) .............................................................................. 16
2.3.1 Carbon limitation .......................................................................................... 16
2.3.2 Interactions between algae and bacteria ......................................................... 19
2.3.3 Carbon cycling by bacteria ............................................................................ 20
2.3.4 Algae-based models ...................................................................................... 21
2.4 Downstream process enhancement by pre-treatment (Part II) ....................................... 23
2.4.1 Lipid extraction and biodiesel production ...................................................... 23
2.4.2 Anaerobic digestion and biomethane production ............................................ 24
3 RESEARCH OBJECTIVES ................................................................................................. 25
3.1.1 Research Objective 1 (RO1) – Understand and model the contribution of bacteria to
algal growth by carbon cycling in lab-scale algal culture system ................................. 25
3.1.2 Research objective 2 (RO2) – Enhance lipid, and specifically triacylglyceride (TAG),
extraction from algae by using free nitrous acid pre-treatment ..................................... 26
3.1.3 Research objective 3 (RO3) – Enhance methane production from algal digestion by
free nitrous acid pre-treatment ..................................................................................... 26
4 REASERCH APPROACH ................................................................................................... 28
4.1.1 Algae and bacteria source .............................................................................. 28
4.1.2 Algal growth and culture conditions .............................................................. 28
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